What are the sample types for protein ab initio sequencing?
Answer: Protein de novo sequencing is independent of the size and length of the protein. Monoclonal antibodies, Fab/Fc, double antibodies, multiple antibodies, recombinant proteins, peptides, fluorescently labeled antibodies, cross-linked antibodies, etc. are available for de novo sequencing.
Can fluorescently labeled or cross-linked antibodies be used for de novo sequencing?
Answer: Proteins cross-linked to beads or fluorescently labeled proteins such as flow-through antibodies FITC, Cyc5, PE and other labeled antibodies are also capable of performing de novo sequencing.
For unknown proteins, how can the protein sequence be accurately analyzed if there is cyclization closure at the N-terminus of the protein or when there is an unknown modification at the N-terminus?
Answer: For proteins with unknown sequences, especially modified recombinant proteins, in vitro synthesized proteins, and modified antibodies, or proteins with unstudied gene sequences, the information of such proteins is often not included in the protein database. Accurate analysis of the sequences of such proteins must be achieved by de novo sequencing of proteins.
What are the requirements for sending samples for protein de novo sequencing?
Answer: The standard sample size is 100ug and the purity is 90% or more. Protein purity has a large impact on sequencing results. Impurity antibody protein, homologous protein and BSA interfered with sample sequencing, thus affecting the experimental results.
Does the sample buffer composition have any effect on the sequencing results?
Answer: The standard sample delivery recommendation for proteolytic buffer is to use PBS or Tris buffer system. If there are glycerol, BSA, descaler, salt, etc., we will do some purification process accordingly, so it will not affect the sequencing results.
Will sample glycosylation and post-translational modifications affect sequencing?
Answer: No. Possible post-translational and glycosylation modifications are taken into account during data analysis and do not affect the sequencing results of the sample itself.
What is the accuracy of the protein de novo sequencing results?
Answer: Sequencing results ensure full sequence coverage and 100% sequence accuracy. Each amino acid at each site is supported by more than ten different peptides. Each amino acid has strong peak evidence of fragmentation in the mass spectra. Sequencing results are combined with software algorithms and manual checks to ensure sequence accuracy.
